Beta-amyloid peptide (Abeta or BetaA4) is the major component in amyloid plaques found in Alzheimer's Disease (AD). Variation in the position of cleavage at the C-terminal end of Abeta from the precursor define the amyloid promoting ability and the neurotoxic potential of the Abeta formed. All of the C-terminal peptide sites, cleaved by the protease termed gamma-secretase, are buried within the membrane bilayer. Recent findings have identified a unifying feature in the physiology of all the known familial, hereditary forms of AD: over-production of the longer, more amyloidogenic and toxic forms of Abeta. This observation underscores the importance of gamma-secretase in the pathophysiology of AD. We have developed a model system for studying the rare gamma-secretase cleavage event in cultured cells that over-express the substrate for the protease, an analogue of the C-99 proteolytic fragment of APP (C/1-102). This system allow quantitation of the substrate and both products of proteolytic cleavage. The proteolytic activity has been localized to the ER or early golgi, in agreement with independent lines of evidence from other laboratories. The cleavage site corresponds to the formation of very long froms of Abeta (up to Abeta/45). Preliminary results indicate that the gamma-secretase activity acts upon a membrane-bound substrate leaving at least one membrane associated product. This result suggests an unprecedented model in which gamma-secretase cleaves an amino acid domain that is ordinarily buried within the lipid bilayer without separating the membrane. We propose, first, to confirm the membrane association of the precursor and product. We will characterize the nature of the association by several experimental approaches in order to understand the orientation of substrate during proteolytic cleavage. We will also examine the membrane association requirements of the gamma-secretase proteolytic activity(ies) and whether intracellular Abeta is membrane associated under conditions where it is formed but not released from cells. We will also characterize the C-terminal sequence of the Abeta secreted by cells over-producing C/t- 102. Characterization of the exact milieu in which gamma-secretase is active will provide insight into the possible types of physiological or structural changes in cells that lead to altered proteolytic activity or specificity. Clearer definition of the requirements and cellular location of the enzyme activity will advance our long-term effort to identify and purify gamma-secretase, an important therapeutic target for the arrest of AD-associated neurodegeneration.